Analysis and detection of biological molecules typically involve placing a sample onto an immobilizing membrane and then performing steps to detect the presence of or quantitate one or more particular biological molecules in the sample. A sample may be spotted directly onto the immobilizing membrane or transferred from a matrix to the immobilizing membrane by blotting. Such a transfer may be necessary because the matrix can be unsuited for many of the biological or chemical assays known to those skilled in the art. The transfer may be passive or energy-driven, such as by an electric current. Once the sample has been transferred to the membrane, the desired assay can be performed on the immobilized sample.
Methods of transferring biological molecules to immobilizing membranes are known in the art. For example, polynucleotide sequences may be transferred from a gel made of agarose or polyacrylamide to a cellulose-derived or nylon membrane. Similarly, proteins may be transferred from an SDS-polyacrylamide gel to a cellulose-derived or nylon membrane. Immobilizing membranes made from nylon or cellulose-derived materials are porous and permit the transfer of polynucleotides or proteins through a variety of processes, some of which are energy independent and some of which, such as electroblotting, are energy-driven.
Many assays performed on biological molecules can be performed on a miniaturized scale. Many of these assays use samples and reagents that oftentimes are expensive or difficult to obtain. Accordingly, assays performed on a miniaturized scale are desirable because they may dramatically reduce the amount of sample and reagents required for performing the assay. Miniaturized assays are especially desired when an expensive or limited sample can be concentrated, thereby reducing the amount of the sample required for the assay while simultaneously increasing the sensitivity, accuracy or efficiency of the assay. In addition to the reduction of volume, miniaturization allows hundreds or thousands of assays to be performed simultaneously.
A heat-shrinkable film such as that reported in International Publication No. WO 99/53319, published Oct. 21, 1999, permits samples to be concentrated for miniaturized assays. What is needed is a laminate including a shrinkable film that can be used to immobilize molecules transferred to the laminate for subsequent detection or assay.
The present invention provides a laminate having an ionic surface that can be used to immobilize sample molecules transferred to the laminate. The laminate includes a shrinkable substrate such as a polyethylene shrink film. The laminate also includes an ionic coating layer. The ionic coating layer may include, for example, one or more ionic polymers, a hydrogel including hydrolyzed azlactone moieties, bifunctional molecules affixed to a hydrogel, or a hydrogel with an overcoating of one or more ionic polymers. The laminate may also include one or mask layers affixed, directly or indirectly to the substrate. Sample molecules may be transferred from a matrix, such as a gel for separating sample molecules, to the laminate by an energy-independent process or by a process that is energy-dependent, such as electroblotting. The ionic surface reversibly affixes desired sample molecules to the laminate. Because the laminate is shrinkable, sample molecules that have been transferred to the laminate may be concentrated for use in a miniaturized assay.
Various other features and advantages of the present invention should become readily apparent with reference to the following detailed description, examples, claims and appended drawings. In several places throughout the specification, guidance is provided through lists of examples. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
For purposes of this invention, the following definitions shall have the meanings set forth.
xe2x80x9cAxe2x80x9d or xe2x80x9canxe2x80x9d refers to one or more of the recited elements.
xe2x80x9cAffixxe2x80x9d shall include any mode of attaching biological molecules to a substrate. Such modes shall include, without limitation, covalent bonding, ionic bonding, and adherence, such as with an adhesive, physical entrapment, and adsorption. This may or may not require the use of linking agents.
xe2x80x9cAmphotericxe2x80x9d as used herein shall mean, with respect to any molecule, compound, composition or complex, having character of both an acid and a base. The term includes molecules, compounds, compositions or complexes that are both anionic and cationic, e.g., a polypeptide at its isoelectric point.
xe2x80x9cBifunctionalxe2x80x9d as used herein shall mean, with respect to any molecule, compound, composition or complex, having more than one functional group. For example, a bifunctional molecule may have an amino group capable of forming a covalent bond with an azlactone moiety and an anionic group capable of forming an ionic bond with a cation.
xe2x80x9cDensityxe2x80x9d shall mean a measure of quantity per unit projected area of a substrate, such as, for example, molecules per square centimeter.
xe2x80x9cHeat-relaxablexe2x80x9d or xe2x80x9cheat-shrinkablexe2x80x9d shall mean, in the context of a material such as a substrate, that the material undergoes some relaxation or shrinkage in at least one dimension in response to the transmission of thermal energy into the material.
xe2x80x9cIonicxe2x80x9d shall mean any chemical species that has a formal charge, i.e., has an excess (negative formal charge) or a deficiency (positive formal charge) of electrons on at least one atom of the species. A polymeric surface is xe2x80x9cionicxe2x80x9d if it contains at least one chemical species having a formal charge even if the polymeric coating is associated with a counterion (e.g., in solution) having an opposite formal charge. The counterion may produce a surface with a net neutral charge even though the polymer surface itself has a formal positive or negative charge.
xe2x80x9cLinking agentxe2x80x9d shall mean any chemical species capable of affixing a xe2x80x9cMoleculexe2x80x9d to a substrate. Linking agents can be covalently bonded to the substrate or can be provided by a polymeric coating thereon.
xe2x80x9cMoleculexe2x80x9d shall be construed broadly to mean any molecule, compound, composition or complex, either naturally occurring or synthesized, that can be detected or measured in or separated from a sample of interest. Molecules include, without limitation, polypeptides, fatty acids, polynucleotides, carbohydrates, polysaccharides, hormones, steroids, lipids, vitamins, bacteria, viruses, pharmaceuticals, and metabolites.
xe2x80x9cPolynucleotidexe2x80x9d shall mean any polymer of nucleotides without regard to its length. Thus, for example, ribonucleotides and deoxyribonucleotides are each included in the definition of polynucleotide as used herein, whether in single- or double-stranded form. A polynucleotide, as used herein, may be obtained directly from a natural source or may be synthesized using recombinant, enzymatic or chemical techniques. A polynucleotide may be linear or circular in topology and can be, for example, a vector such as an expression vector, cloning vector or any type of plasmid, or any fragment thereof.
xe2x80x9cPolypeptidexe2x80x9d shall mean any polymer of amino acids without regard to its length. Thus, for example, the terms peptide, oligopeptide, protein, enzyme, and fragments thereof are all included within the definition of polypeptide as used herein. The term also includes polypeptides that have been modified by post-translational expression or synthetic processes yielding, for example, glycosylated, acetylated, phosphorylated polypeptides, or peptide nucleic acids. Accordingly, a polypeptide may be obtained directly from a natural source or may be synthesized using enzymatic or chemical techniques.
xe2x80x9cPolysaccharidexe2x80x9d shall mean any polymer of saccharides without regard to its size. The term also includes classes of molecules that are polymers of saccharides in combination with other monomers such as amino acids, nucleotides, and any polymers thereof. Such classes of molecules include, but are not limited to, glycosaminoglycans, proteoglycans and glycolipids.
xe2x80x9cProjected surface areaxe2x80x9d shall mean the surface area for a surface as is calculated with respect to the plane encompassing the xe2x80x9cxxe2x80x9d and xe2x80x9cyxe2x80x9d axes of the surface.
xe2x80x9cRecoverablexe2x80x9d means, in the context of a material, such as a substrate, that the material is stretched and capable of subsequently recovering at least one dimension, preferably to substantially its original size.
xe2x80x9cRelaxablexe2x80x9d shall mean, in the context of a material such as a substrate, that the material is capable of relaxing or shrinking, in at least one dimension. Preferably, shrinkage occurs by at least about 10%.
xe2x80x9cShrinkable,xe2x80x9d xe2x80x9cshrinkingxe2x80x9d or xe2x80x9cshrunkxe2x80x9d shall mean, in the context of a material such as a substrate, that the material is capable of being, is, or has been decreased in its length in at least one dimension, whether by recovery, relaxation, or any other means.
xe2x80x9cTopographical surface areaxe2x80x9d shall mean the area of a surface as calculated with respect to the planes encompassing the xe2x80x9cxxe2x80x9d, xe2x80x9cyxe2x80x9d and xe2x80x9czxe2x80x9d axes of the surface.
xe2x80x9cUndulationsxe2x80x9d or xe2x80x9cundulatedxe2x80x9d shall mean convoluted, wave-like forms. For purposes of this invention, it is preferred that an undulated surface includes undulations that do not form a regular pattern. xe2x80x9cUndulationsxe2x80x9d or xe2x80x9cundulatedxe2x80x9d does not include structures such as reservoirs or microwells that are created by methods such as for example printing, embossing, casting, molding, laserscribing, photolithography, etching, mechanical scratching, or scoring.